CN111537451A

CN111537451A - 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of tacrolimus detection reagent

Info

Publication number: CN111537451A
Application number: CN202010009570.2A
Authority: CN
Inventors: 张永侠; 封建新; 李薇; 张启飞; 龚俊; 王贵利; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2020-01-06
Publication date: 2020-08-14
Anticipated expiration: 2040-01-06
Also published as: CN117054643A; CN116718764A; CN116124721A; CN111650135A; CN112285037A; CN116338215A; CN116840467A; CN111504920A; CN116718761A; CN116626281A; CN116735512A; CN116148198A; CN117030640A; CN112285038A; CN111537452B; CN116359146A; CN116699122A; CN116773795A; CN111487207A; CN116008201A

Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a tacrolimus detection reagent. Specifically, the glucose-6-phosphate dehydrogenase mutant of the present application comprises one or a combination of mutations selected from the group consisting of: D306C, D375C, G426C. The detection kit prepared by using the glucose-6-phosphate dehydrogenase mutant has the advantages of strong specificity, high sensitivity, convenient operation, short detection time and accurate quantification, and is suitable for high-throughput detection.

Description

6-phosphoglucose dehydrogenase mutant and application thereof in preparation of tacrolimus detection reagent

Priority of application No. 201910017764.4 filed on 1/9/2019 and 201910423122.4 "mutant glucose-6-phosphate dehydrogenase and use thereof in the preparation of test agents" filed on 5/21/2019, which are incorporated herein by reference.

Technical Field

The application relates to the field of biological detection, in particular to mutant enzyme 6-phosphoglucose dehydrogenase (G6 PDH for short) and application thereof in a tacrolimus detection kit.

Background

Haptens, some small molecular substances (molecular weight less than 4000Da), alone cannot induce an immune response, i.e. are not immunogenic, but can acquire immunogenicity when crosslinked or conjugated with carriers such as macromolecular proteins or non-antigenic polylysine, and induce an immune response. These small molecule substances can bind to response effector products, have antigenicity, are immunoreactive only and are not immunogenic, and are also called incomplete antigens.

The hapten can be combined with a corresponding antibody to generate an antigen-antibody reaction, and can not singly stimulate the human or animal body to generate the antigen of the antibody. It is immunoreactive only, has no immunogenicity, and is also called incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically bound to a protein molecule (carrier), it will acquire new immunogenicity and will stimulate the production of corresponding antibodies in animals.

Small molecule antigens or haptens lack two or more sites that can be used in sandwich assays, and therefore cannot be measured using the double antibody sandwich assay, and often use a competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete to bind with the solid-phase antibody. The more the amount of the antigen in the specimen is, the less the enzyme-labeled antigen bound to the solid phase is, and the lighter the color develops. ELISA measurement of small molecule hormone, medicine, etc. is used in different methods.

Tacrolimus (tacrolimus) is a specific example of a hapten, and the structural formula is shown as follows:

tacrolimus, also known as FK506, is a macrolide antibiotic. Tacrolimus was discovered in japan in 1984, and was first used clinically as an immunosuppressant in 1989. Tacrolimus, as an immunosuppressant, is highly lipophilic, incompletely absorbed and unstable.

The safe and effective therapeutic range of tacrolimus is narrow, and insufficient dosage or low blood concentration of tacrolimus may cause graft rejection. Too high a concentration of tacrolimus can cause serious adverse reactions, including nephrotoxicity, neurotoxicity, post-transplantation diabetes, increased susceptibility to infection, canceration, hypertension, gastrointestinal dysfunction and the like.

For the reasons, tacrolimus blood concentration monitoring is an effective mode for assisting clinical treatment, improving treatment effect and reducing toxicity risk.

The currently known tacrolimus detection methods mainly comprise: high Performance Liquid Chromatography (HPLC), luminescence immunity, enzyme-linked immunosorbent assay (ELISA), etc. The HPLC method needs complex sample pretreatment, has complex operation and long period and is expensive in cost; the reagent of the luminescence immunoassay method has high cost, is not suitable for the detection of conventional treatment drugs, and is not more beneficial to large-scale popularization.

The existing homogeneous enzyme immunoassay method and latex agglutination turbidimetry method are limited in application due to complex preparation process and large batch difference.

The prior art CN108107200A describes a tacrolimus detection kit, which discloses a preparation method of a conjugate of glucose-6-phosphate dehydrogenase and tacrolimus:

dissolving 20-100mg of tacrolimus in methanol, adding anhydrous sodium acetate, adding carboxymethyl hydroxylamine after uniform dissolution, uniformly mixing, heating under the protection of nitrogen for reaction overnight, then carrying out reduced pressure distillation to obtain a wax-like substance, adding dimethylformamide for dissolution, filtering to remove precipitates, and carrying out reduced pressure distillation to remove a solvent to obtain a product A;

-dissolving 10-50mg of product a in 20-100mL of dimethylformamide, and slowly adding 50-150 μ L of carbodiimide (EDC) dropwise to the solution under stirring, and mixing by rotation for 60-150 minutes;

10-50mg of 100-300KU glucose-6-phosphate dehydrogenase is dissolved in PBS buffer and shaken uniformly;

slowly adding the tacrolimus solution into the solution 3 under stirring, and reacting for 8-16 hours under stirring to obtain the 6-phosphoglucose dehydrogenase and tacrolimus conjugate.

However, the prior art methods rely on the activation of a reactive group carried by the small molecule drug (tacrolimus) itself, followed by reaction with an enzyme. Such a conjugation method may result in the situation where multiple tacrolimus are attached to the same glucose-hexametaphosphate dehydrogenase, and it is difficult to ensure consistency of conjugation sites, orientation between small molecule drugs and enzymes 1: 1, resulting in large batch-to-batch variation.

Disclosure of Invention

In view of the need in the art, the present application provides a novel glucose-6-phosphate dehydrogenase mutant and its use in the preparation of tacrolimus detection kits.

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In contrast to the previously published mutant of glucose-6-phosphate dehydrogenase of patent US006090567A (halogenated immunological systems using mutant glucose-6-phosphate dehydrogenes), the glucose-6-phosphate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of: D306C, G426C, D375C.

According to some embodiments, there is provided a glucose-6-phosphate dehydrogenase mutant, the glucose-6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No. 4.

According to some embodiments, there is provided a polynucleotide encoding a glucose-6-phosphate dehydrogenase mutant of the present application.

According to some embodiments, there is provided an expression vector comprising a polynucleotide of the present application.

According to some embodiments, there is provided a host cell comprising an expression vector of the present application. The host cell may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast).

According to some embodiments, there is provided a conjugate of a glucose-6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1: x is coupled.

In some embodiments, x is 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

In some specific embodiments, the glucose-6-phosphate dehydrogenase mutant of the present application is preferably present in a molar ratio of 1: 1.

in some specific embodiments, the hapten has a molecular weight of from 100Da to 4000Da, for example: 100. 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

According to the present application, the skilled person will understand that "hapten" also comprises forms of its derivatives. To facilitate conjugation to glucose-6-phosphate dehydrogenase, haptens (e.g., tacrolimus) that do not themselves bear a coupling group (e.g., a group that reacts with a sulfhydryl group) can be engineered to bear a linker for covalent binding to a sulfhydryl group. Thus, in the present application, a hapten derivative refers to a hapten which has been engineered to carry a thiol-reactive group.

The hapten is selected from: small molecule drugs (e.g. antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids.

Haptens are exemplified by, but not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin, digoxigenin), mycophenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the metabolites cotinine of uretonidine, morphine, derivatives of urodihydroxyphenol, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental prolactin, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuresis hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, free thyroxine, free triiodothyronine, cortisol, Urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfates, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 α hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-keto prostaglandin F1 α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, nalin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, substance P, neurotensin, thromboxane a2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

In a particular embodiment, the hapten is tacrolimus or a derivative thereof.

In particular embodiments, the hapten is a tacrolimus derivative bearing a thiol-reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine.

In a particular embodiment, the hapten is a tacrolimus derivative, as shown in formula I:

in some embodiments, m is an integer from 1 to 10, preferably from 1 to 6, such as 1, 2, 3, 4, 5, 6.

According to some embodiments, there is provided a reagent comprising a conjugate of the present application.

According to some embodiments, there is provided a use of a glucose-6-phosphate dehydrogenase mutant of the present application in the preparation of a tacrolimus detection reagent.

According to some embodiments, there is provided a use of a conjugate of the present application for the preparation of a tacrolimus detection reagent.

In specific embodiments, the detection reagent is selected from the group consisting of: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.

In a specific embodiment, the detection reagent is preferably a reagent for detection based on a competition method.

According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a tacrolimus detection device.

In particular embodiments, the detection device may be prepared in the form of a well plate (e.g., a 96-well plate), such as a plate coated with a reagent according to the present application.

In particular embodiments, the detection device may be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with a reagent according to the present application.

According to some embodiments, there is provided a tacrolimus detection kit comprising:

-a first reagent comprising a substrate, a buffer and a tacrolimus antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;

-a second agent comprising a conjugate of the present application and a buffer;

-optionally, a calibrator comprising 10mM to 500mM buffer, 0ng/ml to 30ng/ml tacrolimus; and

-optionally, a quality control comprising 10mM to 500mM buffer, 5ng/ml to 25ng/ml tacrolimus.

According to one embodiment, there is provided a tacrolimus detection kit comprising:

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 50mM substrate,

0.1 to 10 mug/ml mg/L of tacrolimus antibody,

0.1 to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer solution,

0.1. mu.g/ml to 10. mu.g/ml of a conjugate according to the application,

0.1 to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

a third reagent: which comprises the following steps: the volume ratio of methanol to ethanol is 3: 1, and 0.5-5% of zinc sulfate.

In some embodiments, the buffer is selected from one or a combination of: TAPS, tromethamine buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citric acid-sodium citrate buffer solution, barbital buffer solution, glycine buffer solution, borate buffer solution and trimethylolmethane buffer solution; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 50 to 100 mM; the pH of the buffer is 7 to 8.

In some embodiments, the stabilizing agent is selected from one or a combination of: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; bovine serum albumin is preferred.

In some embodiments, the surfactant is selected from one or a combination of: brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20.

In some embodiments, the preservative is selected from one or a combination of: azide, MIT, biological preservative PC (e.g. PC-300), thimerosal; the azide is selected from: sodium azide and lithium azide.

In some embodiments, the substrate comprises: 6-phosphoglucose, beta-nicotinamide adenine dinucleotide.

In some specific embodiments, the tacrolimus antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, human.

In some specific embodiments, the tacrolimus antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, antigen-binding fragments.

According to some embodiments, there is provided a method of preparing a conjugate comprising the steps of:

1) providing a tacrolimus derivative according to the present application, in particular in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethylsulfoxide);

2) providing a glucose-6-phosphate dehydrogenase mutant, preferably in a buffer (which provides a reaction environment, such as, but not limited to, PBS, Tris, TAPS, TAPSO, buffer pH between 6.0 and 8.0);

3) contacting the tacrolimus derivative and the glucose dehydrogenase-6-phosphate mutant at a molar ratio n:1 for 1 hour to 4 hours (preferably 2 hours to 3 hours) at 18 ℃ to 28 ℃ so as to couple the tacrolimus derivative and the glucose dehydrogenase-6-phosphate mutant to obtain a conjugate;

4) the conjugate is optionally subjected to purification, such as desalting treatment or the like, as required.

In some embodiments, the contacting molar ratio of the enzyme to the hapten in the reaction system is 1: n, wherein n is 1 to 500, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, and ranges between any of the foregoing values; preferably n is 20 to 50.

In some specific embodiments, steps 1) and 2) are interchangeable or concurrent.

In some specific embodiments, the glucose-6-phosphate dehydrogenase comprises one or more free sulfhydryl groups prior to conjugation, thereby allowing for a targeted reaction with tacrolimus.

Wild-type glucose-6-phosphate dehydrogenase does not contain a free sulfhydryl group, and thus in some embodiments, glucose-6-phosphate dehydrogenase is genetically engineered to have a free sulfhydryl group by mutating an amino acid at a specific site (306, 375, or 426) to cysteine.

Drawings

FIG. 1.G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

FIG. 2.G6PDH (D306C) amino acid sequence (SEQ ID No. 2).

FIG. 3.G6PDH (D375C) amino acid sequence (SEQ ID No. 3).

FIG. 4.G6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

Example 1 Synthesis of Tacrolimus derivatives

Wherein m is 1.

Tacrolimus (100mg, 0.11mmol) was added to a round bottom flask, dissolved in dry DCM (5mL), to which was added a catalytic equivalent of 4-N, N-lutidine, DCC (27mg, 0.13mmol) and stirred under nitrogen until all dissolved. 4-Maleimidobutyric acid (20mg, 0.11mmol) was added to the reaction system, and stirred at room temperature (18 ℃ C. to 28 ℃ C., preferably 20 ℃ C. to 25 ℃ C.) for about 4 hours for TLC detection. After the reaction was completed, it was purified directly on a preparative plate (MeOH/DCM ═ 1:20) to finally obtain tacrolimus derivative (51mg, yield 47%).

The structure of the product was confirmed by a conventional method.

This example allows tacrolimus to have a group that can bind to enzymes.

Example 2 coupling of Tacrolimus derivatives to G6PDH molecules

First, the coupling method of the present application

The G6 PDH-tacrolimus conjugate according to the present application, was coupled as follows: a thiol-reactive group (such as but not limited to a maleimide group) on a tacrolimus derivative molecule is covalently bound to a thiol on a G6PDH molecule.

1. The tacrolimus derivative prepared in example 1 was dissolved in N, N-dimethylformamide (10 mg/ml);

g6PDH solution: g6PDH (mutant of the present application or prior art mutant) was dissolved in PB 100mmol, NaCl100mmol, pH 8.0, 5mg/mL enzyme;

3.2 ml of glucose 6 phosphate dehydrogenase mutant solution, 7.5ml of PB solution and 0.5ml of tacrolimus derivative solution are added;

4. the mixed solution is sufficiently shaken at room temperature (18-28 ℃ C., preferably 20-25 ℃ C.) for 2-3 hours, and desalted (desalted solution 100mM PB, 0.1% NaN ₃1% NaCl, pH 8.0), the protein peak was collected, and the resulting product, i.e., G6 PDH-tacrolimus conjugate。

Two, control coupling method

The G6 PDH-tacrolimus conjugate was prepared as disclosed in the example with reference to CN 108107200A:

1. dissolving 20-100mg of tacrolimus in methanol, adding anhydrous sodium acetate, adding carboxymethyl hydroxylamine after uniform dissolution, uniformly mixing, heating under the protection of nitrogen for overnight reaction, then carrying out reduced pressure distillation to obtain a wax-like substance, adding dimethylformamide for dissolution, filtering to remove precipitates, and carrying out reduced pressure distillation to remove a solvent to obtain a product A;

2. dissolving 10-50mg of the product A in 20-100mL of dimethylformamide, slowly and dropwise adding 50-150 mu L of carbodiimide (EDC) into the solution under stirring, and carrying out rotary mixing for 60-150 minutes;

3. dissolving 10-50mg of glucose dehydrogenase with the specification of 100-300KU in PBS buffer solution and shaking uniformly;

4. and (3) slowly adding the tacrolimus solution obtained in the step (2) into the solution obtained in the step (3) under stirring, and reacting for 8-16 hours under stirring.

Example 3 preparation of the kit

A kit for detecting tacrolimus comprising:

reagent R1, comprising:

HEPES buffer 50mM, pH 7.0

10mM glucose-6-phosphate

10mM beta-nicotinamide adenine dinucleotide

1 μ g/ml Tacrolimus antibody (commercially available antibody)

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

reagent R2, comprising:

200mM Tris buffer, pH 8.0

1 ug/ml G6 PDH-tacrolimus conjugate

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

sample extraction liquid: the volume ratio of methanol to ethanol is 3: 1 mixing, and 1% of zinc sulfate;

calibration products: 20mM HEPES buffer, and 0.0, 2.5, 5.0, 10.0, 20.0, 30.0ng/ml tacrolimus (or added as needed);

quality control product: 20mM HEPES buffer, and 8.1ng/ml, 15.4ng/ml, 23.2ng/ml (or added as needed).

And assembling the reagents (optionally containing quality control products and calibration products) into a tacrolimus homogeneous phase enzyme immunoassay kit.

Example of detection

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, an enzyme-labeled antigen (such as G6 PDH-tacrolimus) and a non-labeled antigen (tacrolimus) compete for binding with a quantitative antibody (tacrolimus antibody), when the antibody is more bound with the non-labeled antigen, the more activity released by the enzyme-labeled antigen is, the more NADH is generated by catalyzing a substrate NAD +, and the more absorbance change of NADH is detected at a wavelength of 340nm, so that the content of tacrolimus in the liquid can be calculated.

Completely mixing a human whole blood sample, a quality control product and a calibrator, taking 200 mu l of sample by using a pipette, adding an equal volume of sample extract, immediately covering a cover, oscillating on a vortex oscillator for at least 10 seconds to ensure that the sample is fully mixed, centrifuging on a centrifuge for 5 minutes at a speed of 12000r/min, transferring each supernatant into a small tube, covering the cover tightly, and using the sample for detection.

TABLE 1 parameters of fully automatic biochemical analyzer

Model type	Hitachi 7180 parameter
		Analysis point	[Rate-A][10][25][34]
WAVE(SUB/MAIN)	[410][340]
		S.VIL.	[20]
S.R1	[150]
		S.R3	[50]
ABS.LIMIT:	[32000][ increasing by one]
		CALIB TYPE:	[Spline]
POINT:	[6]SPAN PONIT[6]
		Calibration article	0.0、2.5、5.0、10.0、20.0、30.0ng/ml
Sample(s)	Samples to be tested, e.g. plasma, serum, whole blood, urine, etc

Test example 1 Performance of the kit of the present application

1. Calibration of absorbance

TABLE 2 calibrated absorbances

2. Precision experiment

And measuring high, medium and low quality control products and clinical samples by using the calibration curve established above.

TABLE 3 Total inaccuracy

3. Repeatability of

TABLE 4 Box repeatability

4. Recovering

TABLE 5 recovery

5. Tacrolimus detection kit linearity

TABLE 6 linearity

6. Tacrolimus 37 ℃ reagent accelerated stability

The calibration absorbance is reduced by about 16% after the application reagent is accelerated for 7 days at 37 ℃, and the calibration absorbance is reduced by about 51% after the contrast reagent is accelerated for 7 days at 37 ℃.

TABLE 7.37 ℃ accelerated stability of reagents

Test example 2 antibody inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-tacrolimus conjugate, the activity of G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing NAD to be converted into NADH is reduced, and the difference between an experimental group with the antibody added and an experimental group without the antibody added is compared by detecting the change of NADH amount, wherein the difference is represented by the inhibition capacity of the antibody on G6 PDH.

2. Reaction system

TABLE 8 preparation of reagents for measuring antibody inhibition

3. Results

And comparing the absorbance values of the G6 PDH-tacrolimus conjugate when the antibody is added with the antibody and when the antibody is not added, respectively detecting the absorbance values of the G6 PDH-tacrolimus conjugate to obtain the inhibition situation of the antibody on the G6 PDH.

Compared with the published mutation site (A45C), the mutant of the application has obviously improved antibody inhibition rate which can reach more than 45 percent (G426C: 45 percent; D375C: 57 percent) and can reach 58 percent (D306C). Whereas the inhibition rates of previously published mutation sites (e.g. a45C, K55C) were 41% and 25%.

TABLE 9 antibody inhibition of different G6PDH mutants

While not being bound to a particular theory, it may be partially explained as: compared with the G6PDH mutant (A45C, K55C) in the prior art, the mutation site (i.e. the site for introducing free sulfydryl) in the enzyme mutant of the application is the site for coupling with hapten (such as hormone, small molecule drug and the like). When the hapten binds to a hapten-specific antibody at this position, the steric hindrance formed has minimal effect on the activity of the G6PDH enzyme, and after the introduction of the mutation, it does not substantially affect the steric folding of the molecule. Therefore, the position of this mutation site is very important, and needs to be compatible with the activity of G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

The enzyme mutant has obviously improved antibody inhibition rate. After the conjugate obtained by coupling the enzyme mutant and the tacrolimus is prepared into the kit, the reagent has obvious performance improvement in the aspects of batch variation coefficient, linearity, specificity and the like.

Sequence listing

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Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Cys Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210>3

<211>486

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>VARIANT

<222>(375)..(375)

<223> G6PDH mutant, D at position 375 was replaced with C compared to wild type

<400>3

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Cys Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210>4

<211>486

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>VARIANT

<222>(426)..(426)

<223> G6PDH mutant, G at position 426 was replaced with C compared to wild type

<400>5

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Cys Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

Claims

1. A conjugate of a glucose-6-phosphate dehydrogenase mutant and a hapten in a molar ratio of 1: 1 is formed by covalent coupling;

the hapten is tacrolimus or a derivative thereof;

preferably, the tacrolimus derivative is represented by formula I:

wherein the content of the first and second substances,

m is an integer of 1 to 10, preferably 1 to 6.

2. The conjugate of claim 1, wherein:

the glucose-6-phosphate dehydrogenase mutant comprises a mutation selected from the group consisting of: D306C, D375C, G426C;

preferably, the glucose-6-phosphate dehydrogenase mutant is represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

3. A reagent comprising the conjugate of claim 1 or 2.

4. Use of a conjugate according to claim 1 or 2 in the preparation of a detection reagent, wherein:

the detection reagent is a detection reagent of tacrolimus;

preferably, the detection reagent is selected from: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.

5. Use of a conjugate according to claim 1 or 2 for the preparation of a detection device:

the detection device is a detection device for tacrolimus;

the detection device is selected from any one of the following forms: pore plate, particle, chip and test paper;

preferably, the detection device is used for homogeneous immunoassay of tacrolimus.

6. A tacrolimus detection kit comprising:

a first reagent comprising a substrate, a tacrolimus antibody, a buffer;

a second reagent comprising the conjugate of claim 1 or 2, a buffer;

optionally, a calibrator comprising 10mM to 500mM buffer, 0ng/ml to 30ng/ml tacrolimus; and

optionally, a quality control comprising 10mM to 500mM buffer, 5ng/ml to 25ng/ml tacrolimus.

7. The tacrolimus detection kit according to claim 6, comprising:

a first reagent comprising:

10mM to 500mM, preferably 50mM to 200mM buffer, 5mM to 50mM, preferably 10mM to 20mM glucose-6-phosphate, 5mM to 50mM, preferably 10mM to 20mM oxidized β -nicotinamide adenine dinucleotide, 0.1 μ g/ml to 10 μ g/ml, preferably 1 μ g/ml to 5 μ g/ml anti-tacrolimus antibody, 0.1g/L to 5g/L, preferably 1g/L to 5g/L stabilizer, 0.1g/L to 5g/L, preferably 1g/L to 5g/L surfactant, 0.1g/L to 5g/L, preferably 1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM, preferably 50mM to 300mM buffer, 0.1 μ g/ml to 10 μ g/ml, preferably 1 μ g/ml to 5 μ g/ml of the conjugate of claim 1 or 2, 0.1g/L to 5g/L, preferably 1g/L to 5g/L of a stabilizer, 0.1g/L to 5g/L, preferably 1g/L to 5g/L of a surfactant, 0.1g/L to 5g/L, preferably 1g/L to 5g/L of a preservative;

a third reagent comprising:

the volume ratio is 5: 1 to 2: 1 mixed solution of methanol and ethanol, and

0.5 to 5% zinc sulphate by mass/volume;

the buffer is selected from one or a combination of the following: TAPS buffer solution, phosphate buffer solution, glycine buffer solution, Tris buffer solution, boric acid buffer solution, MOPS buffer solution and HEPES buffer solution;

preferably, the buffer of the first reagent is HEPES buffer;

preferably, the buffer of the second reagent is a Tris buffer;

the pH of the buffer is 7 to 8;

the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; preferably bovine serum albumin;

the surfactant is selected from one or a combination of the following: brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20;

the preservative is selected from one or a combination of the following: azide, MIT, biological preservative PC, thimerosal;

preferably, the preservative is selected from the group consisting of: sodium azide, lithium azide and PC-300.

8. The tacrolimus detection kit according to claim 6, comprising:

a first reagent comprising:

HEPES buffer 50mM, pH 7.0,

10mM glucose-6-phosphate,

10mM oxidized beta-nicotinamide adenine dinucleotide,

1 mu g/ml tacrolimus antibody,

1g/L bovine serum albumin,

1g/L Tween20、

1g/L sodium azide;

a second reagent comprising:

200mM Tris buffer, pH 8.0,

1 μ g/ml of the conjugate of claim 1 or 2,

1g/L bovine serum albumin,

1g/L Tween20、

1g/L sodium azide;

a third reagent comprising:

the volume ratio is 3: 1 a mixed solution of methanol and ethanol, and

0.5 to 5% zinc sulphate by mass/volume.

9. A method of preparing a conjugate comprising the steps of:

1) providing tacrolimus or a derivative thereof;

2) providing a glucose-6-phosphate dehydrogenase mutant as defined in claim 2;

3) the glucose-6-phosphate dehydrogenase mutant is coupled with the tacrolimus or the derivative thereof;

the tacrolimus derivative is shown as a formula I:

wherein the content of the first and second substances,

m is an integer from 1 to 10, preferably an integer from 1 to 6;

preferably, the method for preparing the conjugate comprises the following steps:

1) providing a tacrolimus derivative, preferably in an aprotic solvent;

2) providing a glucose-6-phosphate dehydrogenase mutant as defined in claim 2, preferably in a buffer;

3) contacting the glucose-6-phosphate dehydrogenase mutant and the tacrolimus derivative at 18 ℃ to 28 ℃, preferably 20 ℃ to 25 ℃, for 1 hour to 4 hours, preferably 2 hours to 3 hours, so that the tacrolimus derivative and the glucose-6-phosphate dehydrogenase mutant are coupled to obtain the conjugate;

4) optionally, purifying the conjugate, preferably desalting the conjugate;

step 1) and step 2) are interchangeable or parallel;

the buffer is selected from: PBS, Tris, TAPS, TAPSO,

the pH of the buffer is 6.0 to 8.0;

the aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, dimethyl sulfoxide;

preferably, prior to step 3), said glucose-6-phosphate dehydrogenase mutant comprises a free thiol group; more preferably, the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306, 375, or 426;

preferably, the tacrolimus derivative and the glucose-6-phosphate dehydrogenase mutant are contacted at a molar ratio of n:1, n being from 1 to 50, preferably 10, 15, 20, 25, 30, 35, 40, 45 or 50;

more preferably, n is 30.